Summary The goal of this grant is to understand the role of SHH signaling in regulation of regenerative healing in mammalian skin wounds. Most mammals including humans are deficient in regenerative skin healing. After injury, wound healing typically results in 'repair' (also defined as fibrosis/scarring). This process fails to fully restore the lost tissue to its original form, resulting in ?a cellular band-aid?. The inability of mammals to regenerate normal skin is most notably illustrated by the lack of skin appendages including hair follicles in the wound scar. A major hurdle in hair follicle regeneration in skin wounds is the failure to reestablish the hair follicle inductive niche known as the dermal papilla (DP). Here, we show that SHH activation within 'repaired' (scarring) wound dermis induces de novo DP formation leading to HFN. These data clearly demonstrate that myofibroblasts within scarring wounds are not intrinsically incapable of forming the regenerative niche. We hypothesize that scarring cells may possess the regenerative potential but are missing the crucial developmental cue, SHH. To address this hypothesis, first, we will ask if Wnt signaling, which is active in fibrotic wounds, is required for SHH-driven DP formation and resultant HFN in wounds. For this we will perform loss-of-function studies on b-catenin, an essential effector of canonical Wnt signaling, in genetically modified wounds with ectopic SHH activation (Aim1). We will also examine the potential of mature Wnt-active fibroblasts to convert to DP in Hh-activated wounds after healing is complete. These experiments will address the potential plasticity of fully differentiated cells and how induction by the Hh pathway may alter their phenotype (Aim 1). Second, preliminary single cell RNAseq analyses comparing Hh-activated and WT wounds have identified downstream expression of several BMPs following Hh activation. We will examine the role of BMP signaling in mediating the function of Hh signaling through loss- and gain-of-function studies on BMP receptor signaling. We will also examine the ability of immediate Hh regulators Gli1 and 2, to directly bind BMP gene regulatory sites by Chip-Seq analysis (Aim 2). Third, human wounds strongly resemble 'repaired' murine wounds in many traits. We will test whether human wounds can respond to SHH signals to produce DP by introducing viral active-SmoM2 or soluble SHH agonists into xenograft-and-wounding models. Importantly, we will delineate the response of human dermal cells to SHH through single cell RNAseq analyses, and also compare these results with those from murine wounds (Aim3). These combined studies will advance our understanding of the mechanisms required to induce hair follicle regeneration in an otherwise hairless wound environment and will provide essential new information about how human wounds may compare with murine wounds and be manipulated for improved healing.